HIV-1 Nef enhances the infectivity of cell-free virions by an incompletely understood process. The research proposed here is designed to continue an ongoing investigation of how Nef enhances viral infectivity. Preliminary data indicate that endosomal coat proteins termed adaptor protein (AP) complexes are key co-factors for the modulation of infectivity by Nef. The data also indicate that Nef affects viral entry into target cells, but this effect is not clearly attributable to an influence of Nef on the trafficking of Env. Finally, the data indicate that Nef is present at the plasma membrane within clathrin-coated pits. Overall, these data suggest that Nef modulates a currently unidentified transmembrane protein (or proteins) to enhance viral infectivity. The experiments proposed herein are designed to test the role of the plasma membrane associated adaptor protein complex AP-2 in the enhancement of viral infectivity by Nef; to identify the host cell protein(s) whose modulation is the basis of infectivity-enhancement; and to define the role of these host proteins in viral entry and replication rate. Three specific aims are proposed to achieve these goals: 1) to determine whether AP-2 is a key Nef co-factor for the enhancement of viral infectivity using RNA interference and the expression of dominant negative proteins; 2) to identify and correlate specific changes induced by Nef in the host cell plasma membrane- and virion-proteomes; and 3) to validate candidate cellular proteins as mediators of Nef's virologic effects using RNA interference and protein overexpression during in vitro assays of viral infectivity, entry, and replication rate.